miR-6883 downregulates HIF1α in colorectal and breast cancer cells

Colorectal cancer (CRC) and breast cancer (BC) are deadly diseases that rank as the second and fourth leading causes of cancer-related deaths, respectively. We have previously shown that miR-6883 targets CDK4/6 and that palbociclib-mediated CDK4/6 inhibition destabilizes HIF1α. We hypothesize that miR-6883 downregulates HIF1α in CRC and BC cells. miR-6883 was transfected into cells under normoxia or hypoxia and western blot analysis revealed that miR-6883 downregulates CDK4/6 and HIF1α in CRC and BC cells, pointing to miR-6883 as a promising therapeutic to target hypoxic tumors or HIF1α-deregulated cancer cells. Future studies will further investigate miR-6883 as a cancer biomarker, effects on HIF–related proteins, and therapeutic uses in vivo .

A) Colorectal cancer cells were treated with doses of palbociclib ranging from 0-20 µM.Cell viability was measured by imaging the bioluminescent signal after addition of CellTiter-Glo reagent.B) Percent viability of colorectal cancer cells treated with palbociclb was calculated and nonlinear regression analysis was completed using GraphPad Prism software.C-H) CRC 1/26/2024 -Open Access cells were transfected with miR-6883 under normoxia or hypoxia (<0.5% O 2 ) and protein levels of CDK4/6, HIF1α, Glut1, and Ran were measured by Western blot.

Description
Colorectal Cancer (CRC) is characterized by the proliferation of abnormal cells in the colon or rectum.CRC originates from small polyps, which are often benign but can become cancerous (American Cancer Society, 2020).In 2023, there were an estimated 106,970 new cases of colon cancer and 46,050 new cases of rectal cancer in the United States, although recently incidence has been decreasing around 1% each year likely due to early screening, fecal immunochemical tests (FIT), and colonoscopies (American Cancer Society, 2023b; Roselló et al., 2019).Mortality is also decreasing; the number of CRCrelated deaths in the United States in 2023 is estimated to be 52,550 compared to 53,200 in 2020(American Cancer Society, 2020;Siegel et al., 2023).However, CRC remains the third most diagnosed cancer and the second leading cause of cancerrelated mortality in the United States (American Cancer Society, 2023b).Colorectal cancer incidence has been rising over the last two decades among younger individuals for unclear reasons (American Cancer Society, 2020).Though treatments for CRC such as surgery, chemotherapy, targeted therapy, immunotherapy, and radiation can help to prolong patients' lives, additional treatment options are needed for this deadly disease (Mayo Clinic, 2023).Excluding skin cancers, breast cancer is the most common cancer in women (American Cancer Society, 2023a) and has a 5-year survival rate of 30% once it spreads to distant organs (American Cancer Society, 2023c).Breast cancer is normally treated with surgery, chemotherapy, hormone therapy, or radiation but new treatments are needed to improve survival outcomes while avoiding treatment-related toxicities (Mayo Clinic, 2022).
Micro-RNAs (miRs) are a group of non-coding RNAs that can control the translation of coding genes by binding to mRNA and preventing its translation (Mehrgou et al., 2021).miRs can bind oncogenes or tumor suppressors to modulate the initiation, progression, metastasis, and recurrence of CRC (To et al., 2018).Current clinical trials are investigating the efficacy of therapeutic miRs in various types of cancer such as lymphoma, mycosis fungoides, melanoma, lung cancer, liver cancer, and myeloma (Menon et al., 2022;O'Neill, 2016;Zhang et al., 2021).
The tumor microenvironment often becomes hypoxic due to rapid oxygen consumption by tumors as well as the formation of atypical tumor blood vessels (Li et al., 2021;Muz et al., 2015).When cancer cells detect a hypoxic environment, hypoxiainducible factor 1 alpha (HIF1α) is stabilized (Cao et al., 2009).Hypoxic conditions and HIF1α signaling have a proangiogenic effect and contribute to the formation of endothelial cells, which improves the delivery of oxygen and nutrients to the tumor (Krock et al., 2011).
First, we confirmed that CRC cells are sensitive to CDK4/6 downregulation.HCT116, HT29, and SW480 cells were treated with increasing doses of the CDK4/6 inhibitor palbociclib for 72 hours and a CellTiter-Glo assay was used to measure cell viability.Bioluminescent imaging (panel A) and analysis in GraphPad Prism (panel B) revealed that CRC cells are sensitive to palbociclib (IC50 = ~8 µM), providing a rationale to test the CDK4/6-targeting miR-6883 as a potential therapeutic agent for this disease.As CDK4/6 inhibitors are already used to treat patients with BC, it was not necessary to confirm their sensitivity to palbociclib.

CellTiterGlo Assay
CRC cells were plated at a density of 5,000 cells per well of a 96-well plate.Cells were treated with palbociclib using doses ranging from 0-20 μM.Cell viability was measured by adding CellTiter-Glo reagent followed by bioluminescence imaging.

Western Blot
Cells were harvested and lysed using a RIPA buffer containing a protease inhibitor.Denaturing sample buffer was added, samples were boiled at 95°C for 10 minutes, and an equal amount of protein lysate was electrophoresed through 4-12% SDS-PAGE gels (Invitrogen) then transferred to PVDF membranes.The membrane was blocked with 5% milk in 1 × TTBS, incubated overnight in the appropriate primary antibody (HIF1α, pRb, Glut1, CDK4/6, or Ran), and incubated in the appropriate HRP-conjugated secondary antibody for two hours.The levels of antibody binding were detected using ECL western blotting detection reagent and the Syngene imaging system.

Transfection
CRC cells (HT29, HCT116 and SW480) and BC cells (MCF7, MDA-MB-468 and MDA-MB-231) were plated at various cell densities and incubated for at least 12 hours to allow cells to adhere to the plate.The cells were then transfected with 50 nmol/L miR-6883 mimic (Sigma-Aldrich HMI2616) using Lipofectamine® RNAiMAX Reagent.The cells were incubated for either 48 or 72 hours under normoxia (~20.9%O 2 ) and hypoxia (<0.5% O 2 ).